CN114395487A - Walnut root rot pathogen ML-1, biocontrol microbial inoculum and application thereof - Google Patents

Walnut root rot pathogen ML-1, biocontrol microbial inoculum and application thereof Download PDF

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CN114395487A
CN114395487A CN202210049669.4A CN202210049669A CN114395487A CN 114395487 A CN114395487 A CN 114395487A CN 202210049669 A CN202210049669 A CN 202210049669A CN 114395487 A CN114395487 A CN 114395487A
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root rot
walnut
pathogenic bacteria
pathogen
walnut root
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CN114395487B (en
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辛磊
李秀玲
谢代祖
覃国乐
安慧
莫霖
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Hechi University
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
    • A01N63/22—Bacillus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

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  • Biotechnology (AREA)
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  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Virology (AREA)
  • Microbiology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
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  • Agronomy & Crop Science (AREA)
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  • Pest Control & Pesticides (AREA)
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Abstract

The invention relates to the technical field of microbial application, and particularly discloses a walnut root rot pathogen ML-1 and application thereof, wherein the preservation number of the pathogen ML-1 is CGMCC 3.20697, and the preservation date is 2021, 11 months and 27 days. The pathogenic bacteria ML-1 obtained by the invention is one of the pathogenic bacteria causing the root rot of the local walnut, and the bacillus subtilis has obvious inhibition effect on the pathogenic bacteria.

Description

Walnut root rot pathogen ML-1, biocontrol microbial inoculum and application thereof
Technical Field
The invention relates to the technical field of microbial application, in particular to a walnut root rot pathogen ML-1, a biocontrol microbial inoculum and application thereof.
Background
Stony desertification is a phenomenon of vegetation damage, water and soil loss and land degradation caused by human factors. The Guangxi rocky desertification area reaches more than 3000 ten thousand mu, which is second to Yunnan and Guizhou, and is third in China, and the rocky desertification phenomenon is particularly serious when the river pool city is used as a main area of the karst landform in northwest of Guixi. Serious stony desertification has serious influence on local ecology, social economy and people's life, and the treatment work becomes an important task at present. Due to the special severe conditions of the stony desertification regions, the effect of the treatment project mainly based on forest and grass is very little, and how to effectively repair the stony desertification is the key to protect water and soil resources and prevent water and soil loss.
The walnut (Juglans regia L.) plays an important role in economic tree species in China, is the top of four famous dried fruits in the world and has extremely high economic value. The long history of walnut cultivation in China is one of the world walnut cultivation origins, but the walnut products in China have weak competitiveness in the aspects of the occupancy and the sale price of the international market due to wild planting technology, poor management experience and serious pest and disease damage phenomenon.
River pond city is one of the most severe regions of rock desertification in Guangxi province. The large-scale walnut planting in the stony desertification area in the river pond city from 2012 has been developed to over 260 mu, is the city with the largest walnut planting area in the karst stony desertification area in southwest, and is also the city with the largest walnut planting area in the south China. However, the diseases and insect pests are particularly serious due to various causes such as technology, management, and environment.
Related researches on walnut root rot are few in China, pathogenic bacteria capable of causing the walnut root rot include fusarium, pythium, rhizoctonia, phytophthora, alternaria, helminthosporium and the like, and the fusarium is a main pathogenic bacterium for fungal infection.
Root rot belongs to soil-borne diseases, fungal spores are easy to spread, and the prevention and control difficulty is high. At present, chemical prevention and control are mainly used in China, but the environment is easily polluted.
Disclosure of Invention
In order to solve the technical problems, the invention provides a walnut root rot pathogen ML-1, a biocontrol microbial inoculum and application thereof, wherein the pathogen ML-1 is one of pathogens causing walnut root rot, and bacillus subtilis has an obvious inhibiting effect on the pathogens.
The invention aims to provide a walnut root rot pathogen ML-1, wherein the preservation date of the pathogen ML-1 is 2021, 12 months and 27 days, and the preservation number is CGMCC 3.20697.
Furthermore, a culture medium used for culturing the pathogenic bacteria ML-1 is a fungus culture medium.
Further, the fungus culture medium is a PDA culture medium.
Further, the PDA culture medium formula is as follows: 200g of PDA culture medium, 20g of glucose, 15g of agar and 1L of sterile water.
The second purpose of the invention is to provide a biocontrol microbial inoculum for inhibiting the pathogenic bacteria ML-1.
Further, the biocontrol microbial inoculum comprises bacillus subtilis.
Further, the biocontrol microbial inoculum is prepared from bacillus subtilis.
The third purpose of the invention is to provide the application of the bio-control microbial inoculum in preventing and treating the walnut root rot.
Further, the walnut root rot is triggered by pathogenic bacteria ML-1.
Compared with the prior art, the invention has the beneficial effects that:
1. the invention provides a walnut root rot pathogen ML-1, wherein the pathogen ML-1 is one of pathogens causing walnut root rot, and bacillus subtilis has an obvious inhibiting effect on the pathogens and can be used for preventing and treating the walnut root rot.
Biological material preservation information description
ML-1, referred to as a pathogen ML-1 in the present application, has been deposited at 27.12.2021 in the China general microbiological culture Collection center (CGMCC) with a collection number of CGMCC 3.20697, and the location of the collection unit is the institute of microbiology, China academy of sciences, Ministry of sciences, North road 1, Cheng Xilu, Beijing, the rising area, the postal code: 100101, classified and named as Fusarium equiseti (Fusarium equiseti).
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a strain morphology diagram and an electron microscope scanning diagram of pathogenic bacteria ML-1 obtained by separation in the invention;
wherein A is a strain morphology map of ML-1;
b is an ML-1 electron microscope scanning image;
FIG. 2 is a graph showing the effect of pathogenic bacteria ML-1 on the confrontation of Bacillus subtilis in the present invention.
Detailed Description
The following detailed description of specific embodiments of the invention is provided, but it should be understood that the scope of the invention is not limited to the specific embodiments. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention. The experimental methods described in the examples of the present invention are all conventional methods unless otherwise specified.
The invention provides a walnut root rot pathogen ML-1, a biocontrol microbial inoculum and application thereof.
The specific embodiment is as follows:
example 1
Materials (I) and (II)
1. Experimental culture medium and formula
Screening a culture medium: 200g of potato, 20g of glucose, 15g of agar and 1L of sterile water. Penicillin sodium and streptomycin sulfate.
Fungus culture medium: 200g of PDA culture medium, 20g of glucose, 15g of agar and 1L of sterile water.
Bacteria culture medium: 5g of yeast extract, 10g of tryptone, 10g of sodium chloride, 15g of agar, 950ml of sterile water and 50ml of 5mol/L sodium hydroxide solution.
2. Experimental Material
Pathogenic bacteria screening materials: walnut plant root system tissues with typical diseases are around the river pond city.
Antagonistic bacteria: bacillus subtilis, a laboratory-preserved strain.
Second, Experimental methods
1. Pathogen sample collection
Root tissues of plants withered due to walnut root rot infection are collected in walnut plantations around the river pond city, the cause of death of the plants due to the withered branches and trunks is stored in a refrigerator at 4 ℃ for short term for later use.
2. Isolation of pathogenic fungi
Cutting the root of the collected diseased plant with a scalpel to obtain a cube root block with the side length of 0.5cm, cleaning the cube root block with tap water, putting the cube root block into a culture dish which is arranged in a super-clean workbench and is provided with filter paper, fully absorbing the moisture on the surface of the root block, disinfecting the surface by adopting a three-step method, and cleaning the root block with sterile water after disinfection. And (3) placing the treated root blocks in a culture dish filled with filter paper, fully absorbing the moisture on the surface of the root blocks, inserting the root blocks into a screening culture medium by using forceps, placing the culture medium in a constant-temperature incubator for culture, picking hyphae from the edges of the bacterial colonies by using an inoculating needle, and inoculating the hyphae into a PDA (personal digital assistant) plate for culture for later use.
3. Pathogenic bacteria pathogenicity determination
Activating the selected pathogenic bacteria, pouring sterile water into a culture dish, lightly scraping hypha with an inoculating loop to make conidia of the pathogenic bacteria into bacterial suspension in the sterile water, and diluting the bacterial suspension until the number of the spores is 1 × 10-6One/ml for standby. Cleaning root of healthy walnut seedling with sterile water, sterilizing with 75% alcohol, cutting one side root of walnut seedling with scalpel, and sterilizing with bacteriaSoaking the wound for 2h by using the suspension, then placing the walnut young seedlings in a seedling pot, wherein the soil in the seedling pot is sterile soil, and finally pouring the soaked bacterial suspension at the roots of the walnut seedlings; sterile water was used as a control. After inoculation, the two groups of plants were cultured in the same environment, and the growth of the two groups of plants was recorded. After the experimental group is ill, the pathogenic bacteria are separated again from the walnut seedling root tissue blocks of the ill group, and whether the pathogenic bacteria separated again are consistent with the inoculated pathogenic bacteria or not is observed through comparison.
4. Morphological identification of walnut root rot pathogenic bacteria
After the pathogenic bacteria are activated, the size and the shape of a bacterial colony of the pathogenic bacteria, the shape of hypha and the growth condition of the bacterial colony are observed and recorded by an optical microscope according to Chinese fungus articles of forest dawn people and the like, and the microstructural characteristics of the hypha, conidia and spore peduncles are observed under an electron microscope.
5. Antagonistic bacteria confrontation experiment
Activating pathogenic bacteria and antagonistic bacteria (Bacillus subtilis), and inoculating two strains of walnut root rot pathogenic bacteria in a culture dish with a culture medium and a diameter of 9cm, wherein the distance between the two strains of walnut root rot pathogenic bacteria and the center of the culture dish is 2cm, and the two strains of pathogenic bacteria pass through the center point of the culture dish to form a straight line. Culturing for two days in an incubator, inoculating screened antagonistic bacteria in the centers of two pathogenic bacteria after hypha grows out, placing in the incubator, continuously culturing, and observing the phenomenon.
Third, experimental results
1. Separating and purifying walnut root rot pathogenic bacteria strain
Separating and purifying to obtain a strain of walnut root rot pathogenic bacteria, and recording as ML-1.
2. Pathogenic bacteria pathogenicity experiment of walnut root rot
After the counter-dyed healthy walnut seedlings are cultured for 30 days, the typical symptoms of walnut root rot appear in the test group, and the symptoms of the control group walnut seedlings irrigated by sterile water do not appear. The leaves of the plants in the experimental group turn yellow, and the leaf wilting phenomenon appears at the beginning of some plants, roots are excavated, the roots are found to be black, and parts of the roots begin to rot, and the symptoms are similar to the symptoms of typical walnut root rot. And (3) culturing the root tissue blocks of the diseased plants by using a PDA culture medium, observing the colony morphology of the obtained pathogenic bacteria and the colony morphology of the originally inoculated pathogenic bacteria, finding that the colony morphologies and the growth conditions of the two pathogenic bacteria are the same, and preliminarily proving that the pathogenic bacteria are the walnut root rot pathogenic bacteria in the stony desertification areas of river ponds.
3. Morphological identification
After the ML-1 strain is cultured, the average radius of a bacterial colony is 2.50cm, the color of hypha is white, the shape is flocculent (the left side in figure 1), the bacterial colony has two types of conidia, the shape of a small conidia is oval, the size difference is not large, no separation or a separation (as the right side in figure 1) exists 2-3 days after inoculation, and the average size is 6 multiplied by 8 mu m; the large conidia appear 3-5 days after inoculation, are sickle-shaped, have 1-3 partitions and have small size difference. According to the above morphological observation, the strain can be preliminarily identified as fusarium equiseti.
4. Antagonistic bacteria confrontation experiment
As shown in fig. 2, although the walnut root rot pathogenic bacteria grow hypha first, five days after the antagonistic bacteria are inoculated, a remarkable bacteriostatic zone is formed between the pathogenic bacteria and the antagonistic bacteria, which indicates that the antagonistic bacteria have a good bacteriostatic effect on the pathogenic bacteria, that is, the bacillus subtilis has a inhibitory effect on the ML-1 strain of the application.
While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.

Claims (9)

1. The walnut root rot pathogen ML-1 is characterized in that the preservation date of the pathogen ML-1 is 2021, 12 months and 27 days, and the preservation number is CGMCC 3.20697.
2. The walnut root rot pathogen ML-1 of claim 1, wherein a culture medium used for culturing the pathogen ML-1 is a fungal culture medium.
3. The walnut root rot pathogen ML-1 of claim 2, wherein the fungal culture medium is a PDA culture medium.
4. The walnut root rot pathogen ML-1 of claim 3, wherein the PDA medium formula is: 200g of PDA culture medium, 20g of glucose, 15g of agar and 1L of sterile water.
5. A biocontrol microbial inoculum for inhibiting walnut root rot pathogenic bacteria ML-1 in claim 1.
6. The biocontrol agent of claim 5, wherein said biocontrol agent comprises Bacillus subtilis.
7. The biocontrol agent of claim 6, wherein said biocontrol agent is made from Bacillus subtilis.
8. The use of the biocontrol microbial inoculum of claim 5 in preventing and treating walnut root rot.
9. The use of the biocontrol microbial inoculum of claim 8 in the control of walnut root rot, wherein the walnut root rot is triggered by pathogenic bacteria ML-1.
CN202210049669.4A 2022-01-17 2022-01-17 Walnut root rot pathogen ML-1, biocontrol microbial inoculum and application thereof Active CN114395487B (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114540251A (en) * 2022-03-29 2022-05-27 成都建工预筑科技有限公司 Bacillus and application thereof

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112779184A (en) * 2021-01-15 2021-05-11 河北冀微生物技术有限公司 Bacillus subtilis Z-14 strain for preventing and treating root rot of fruits and vegetables and application thereof

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112779184A (en) * 2021-01-15 2021-05-11 河北冀微生物技术有限公司 Bacillus subtilis Z-14 strain for preventing and treating root rot of fruits and vegetables and application thereof

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
杨剑锋等: "呼和浩特市苜蓿根腐病病原菌鉴定及致病性测定", 植物保护学报, pages 119 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114540251A (en) * 2022-03-29 2022-05-27 成都建工预筑科技有限公司 Bacillus and application thereof

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